| Aquatic water are rich in microbial communities, which have mutualistic relationships with aquatic plants and farm animals. Under certain conditions, some microorganisms become potential pathogenic bacteria under the influence of internal and external factors in the water environment, which can cause bacterial diseases and thus affect the survival and growth of farm animals. Urechis unicinctus is the only Xenopneusta distributed along the coast of China, which has rich nutritional and medicinal values. U. unicinctus is a lowly filter-feeding marine invertebrate that inhabits muddy and sandy substrates and is susceptible to attack by external pathogens. It mainly relies on physical barriers and cellular and humoral immune factors in the coelomic fluid to constitute a non-specific immunity for body defense. The coelomic fluid of U. unicinctus is equivalent to the blood of vertebrates, and the internal organs are bathed in it, which is an important way to play the role of immunity. Currently, studies on the coelomic fluid of U. unicinctus are mainly concerned with the isolation and purification of fibrinolytic enzymes and the effects of environmental factors such as sulfide, hexavalent chromium, and bisphenol A stress, while there are fewer studies on the effects of bacteria on U. unicinctus. In order to probe the antioxidant and immune response pattern of U. unicinctus coelomic fluid in response to bacterial infection, the experiments were conducted. Based on the finding that the coelomic fluid of U. unicinctus has a certain inhibitory effect on aquatic pathogens, the present experiment was carried out to determine the number and phagocytosis rate of coelomocytes, antioxidant and immune indexes of the coelomic fluid, and expression of immune-related genes by bacterial attack on U. unicinctus with the aim of providing a reference to the study of the immune defense mechanism of U. unicinctus to cope with bacterial infections.
In this experiment, Aeromonas veronica and Micrococcus luteus were selected for attack test on U. unicinctus, and coelomic fluid samples were taken at 0.5, 1, 3, 6, 12, and 24 h. In the first place, 100 μL of samples were removed to measure the cell number and phagocytosis rate. The rest of the samples were centrifuged at 3000 r/min for 10 min at 4℃, and the coelomic fluid supernatant and cell precipitates were collected, respectively. The cell precipitate was suspended with sterile saline, and the coelomocytes fragmentation supernatant was collected by centrifugation after fragmentation. The total antioxidant capacity, catalase activity, NO content, and lysozyme activity were determined in the two supernatants, respectively. Meanwhile, freshly obtained coelomic fluid was centrifuged at 3000 r/min for 15 min at 4℃, and the collected coelomocytes precipitate was washed three times with sterile saline, and then the coelomocytes were resuspended with L-15 cell culture medium, and the concentration was adjusted to 1×106 cells/mL to obtain the coelomocytes suspension. Then, the coelomocytes suspension was mixed with A. veronica, M. luteus, and (A. veronica+lipopolysaccharides), respectively. The mixture was taken at 0, 5, 10, 20, 30, 40, 50 and 60 min of treatment, and centrifuged at 1500 r/min for 5 min, and the absorbance value of the supernatant was measured at 404 nm to analyze the hemolytic reaction of coelomocytes. Based on previous transcriptome sequencing of U. unicinctus under Vibrio anguillarum treatment, we screened five immunity-related genes, including cbl-b, gck, actr3, lhpp, and cyp450, and set up a control group and four experimental groups (A. veronica, M. luteus, V. anguillarum, and Staphylococcus aureus), and bacterial attack toxicity test was performed separately on U. unicinctus using sterile saline as a control. Coelomic fluid was collected with a sterile syringe at 3, 6, 12, 24, 48 and 96 h of treatment, respectively. The coelomocytes were obtained by centrifugation at 3000 r/min for 15 min at 4℃. Then, the changes in the expression patterns of immune-related genes in coelomocytes were analyzed by real-time fluorescence quantitative PCR, using the β-actin gene and the 18s rRNA gene as internal reference genes.
The results showed that the number and phagocytosis rate of coelomocytes showed a trend of decreasing, then increasing and then decreasing after different bacterial treatments, and the intensity of hemolytic reaction was in the following order：A. veronica+lipopolysaccharide group＞A. veronica group＞M. luteus group. The total antioxidant capacity, catalase activity and NO content of the coelomic fluid supernatant and coelomocytes fragmentation supernatant decreased, then increased and then decreased again, and the lysozyme activity increased and then decreased and then increased, with the prolongation of the bacterial treatment time. The expression of cbl-b, actr3, gck and lhpp in the coelomocytes of U. unicinctus firstly increased and then decreased, and the expression of cyp450 generally showed a trend of firstly decreasing, then increasing and then decreasing.
In conclusion, different pathogens can significantly affect the number of coelomocytes and coelomocytes phagocytosis rate, as well as the antioxidant capacity and immune indexes of the coelomic fluid in U. unicinctus. The comparison revealed that the antioxidant and immunologic capacities of coelomic fluid supernatant in U. unicinctus were stronger than those of coelomocytes fragmentation supernatant in presence of bacterial stimulation. Lipopolysaccharide induced cellular immunity and improves antioxidant capacity of U. unicinctus. This experiment may provide an insight into the innate immune mechanisms of U. unicinctus in response to bacterial infections. |
